Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 100–106
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Binding of antioxidant flavone isovitexin to human serum albumin investigated by experimental and computational assays Ícaro Putinhon Caruso a,b , Wagner Vilegas c , Fátima Perreira de Souza a,b , Marcelo Andrés Fossey a,b , Marinônio Lopes Cornélio a,b,∗ a Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas (IBILCE), UNESP, Rua Cristovão Colombo 2265, CEP 15054-000, São José do Rio Preto, SP, Brazil b Centro Multiusuário de Inovac¸ão Biomolecular (CMIB), Instituto de Biociências, Letras e Ciências Exatas (IBILCE), UNESP, Rua Cristovão Colombo 2265, CEP 15054-000, São José do Rio Preto, SP, Brazil c Instituto de Química (IQ), UNESP, Rua Prof. Francisco Degni 55, CEP 14800-900, Araraquara, SP, Brazil
a r t i c l e
i n f o
Article history: Received 27 January 2014 Received in revised form 30 April 2014 Accepted 13 May 2014 Available online 22 May 2014 Keywords: Isovitexin Human serum albumin Fluorescence spectroscopy Binding density function Molecular modeling
a b s t r a c t The flavonoids are a large class of polyphenolic compounds which occur naturally in plants where they are widely distributed. Isovitexin (ISO) is a glycosylated flavonoid that exhibits a potential antioxidant activity. Some recent studies have shown the pharmacokinetic activity of isovitexin in rat blood plasma, however, without detailing the molecular target that is linked and what physicochemical forces govern the interaction. In mammalians, the most abundant protein in blood plasma is the albumin and is not unlike with human, which human serum albumin (HSA) is the major extracellular protein and functions as a carrier of various drugs. The interaction between HSA and ISO was investigated using fluorescence, UV–vis absorbance, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) together with, computational methods like ab initio and molecular modeling calculation. Fluorescence quenching indicated that ISO location is within the hydrophobic pocket in subdomain IIA (site 1) of HSA, close to the Trp214 residue. The Stern–Volmer quenching constants determined at 288, 298 and 308 K and its dependence on temperature indicated that the quenching mechanism was static. From the analysis of binding equilibrium were determined; the binding site number and binding constants, with the correspondent thermodynamic parameters, H, G and S for HSA–ISO complex. Also, a second binding analysis, binding density function (BDF) method, which is independent of any binding model pre-established obtained similar results. The fluorescence resonance energy transfer estimated the distance between the donor (HSA–Trp214) and acceptor (ISO), while FT-IR and CD spectroscopy measured possible changes of secondary structure at the formation of the HSA–ISO complex. The optimized geometry of isovitexin calculation performed with its ground state by using DFT/B3LYP/6-311+G(d,p) method. The HSA–ISO complex interactions determined by molecular modeling tool corroborated with the thermodynamic analysis from the experimental data. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Flavonoids are a large class of naturally occurring polyphenolic compounds widely distributed in plants. The literature shows that flavonoids can present antioxidant, anticancer, antiviral,
∗ Corresponding author at: Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas (IBILCE), UNESP, Rua Cristovão Colombo 2265, CEP 15054-000, São José do Rio Preto, SP, Brazil. Tel.: +55 17 32212246; fax: +55 17 32212247. E-mail addresses: [email protected] (Í.P. Caruso), [email protected] (W. Vilegas), [email protected] (F.P. de Souza), [email protected] (M.A. Fossey), [email protected], [email protected] (M.L. Cornélio). http://dx.doi.org/10.1016/j.jpba.2014.05.015 0731-7085/© 2014 Elsevier B.V. All rights reserved.
anti-inflammatory and heart disease protective activities [1]. Isovitexin (apigenin-6-C--d-glucopyranoside) is a glycoside flavonoid consisting of the flavone apigenin and the saccharide glucose, as shown in Fig. 1. Like many antioxidants present anti-inflammatory activity, ISO exhibits a potential antioxidant as well; studies have demonstrated that, in inflammatory processes induced by lipopolysaccharide (LPS) in mouse macrophage, this flavone is capable of inhibiting the production and, or release of tumor necrosis factor ␣ (TNF-␣) and prostaglandin E2 (PG2 ) [2]. Studies also have showed that isovitexin presents a possibly antiulcerogenic activity and it could help in peptic ulcer treatment [3]. ISO molecule is found, for example in rice hulls (Oryza sativa L.) [2], “semprevivas chapadeira” (Syngonanthus bisulcatus Rul.) [3] and tanxiang
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101
HSA and ISO were 4.0 M. The final ethanol concentration in buffer was 0. The negative enthalpic term provided the greatest contribution for G◦ , characterizing an enthalpically driven exothermic reaction. The enthalpy–entropy magnitude balance indicated that the hydrophobic interaction offered the main contribution for the formation and stabilization of the HSA–ISO complex. However, signal values for enthalpy and entropy changes also indicated that the electrostatic interactions could play an important role in complex stabilization by forming interactions as van der Walls, hydrogen bonds and charge neutralization.
Fig. 3. Titrations of isovitexin in HSA solutions, as monitored by fluorescence quenching of the Trp214 residue of HSA for three different protein concentrations at pH 7.0 and 298 K. LT 1 , LT 2 and LT 3 represent the ligand concentration for the constant fractional signal molar change value of three different protein concentration (MT 1 , MT 2 and MT 3 ). The insert corresponds to only three sets of concentration pairs (LT ; i and LF values. MT ) to exemplify the utilization of Eq. (9) obtaining
MTx ) (x = 1, 2 and 3) for which LF and i were constants (see insert in Fig. 3). i values obtained from Eq. (6), it can be build From the LF and the Scatchard plot for the binding between HSA and ISO (Supplementary material, Fig. S5) without using any binding model a priori. Scatchard plot was built using the following equation:
i
LF
= n Ka − Ka
i
(8)
where Ka is the association constant and n is binding site number. The obtained values for Ka and n from Scatchard plot were 2.35 × 105 M−1 and 1.04, respectively, which are in accordance with the results obtained from analysis of binding equilibria (Section 3.2). 3.5. Distance measurement between HSA–Trp214 and isovitexin
3.4. Binding density function method The thermodynamic basis for the binding density function (BDF) of ligands bound per macromolecule method is that the distribution in i different states ( i ) is strictly determined at equilibrium by the free ligand concentration (LF ). Thus, if LF is the same for two (or more) solutions at different total macromolecule concentration i will also be the same in each solution. As a result, (MT ), then constant values of LF and i will exist for a number of different combinations of LT and MT , that satisfy the mass conservation equation [21]:
Fluorescence resonance energy transfer (FRET) is a method that can monitor the proximity and relative angular orientation of fluorophores. According to Förster non-radiative energy transfer theory. The energy transfer occurs under the following conditions. The donor produces fluorescent light, the emission spectrum of the donor and the absorbance spectrum of the acceptor have a partial overlap, and the distance between the donor and acceptor is less than 8.0 nm [18]. According to this theory, the average distance r in the binding between HSA–Trp214 (donor) and ISO (acceptor) can be calculated by the equation:
LT = LF +
E =1−
i MT
(6)
Considering the general relationship between the concentration of each macromolecule species with i ligands bound and the experimentally observed signal (Sobs ) from the macromolecule, the fractional signal molar changes (Sobs ) observed in the presence of LT and MT will be given by: Sobs
S − SF MT = obs SF MT
(7)
where SF MT is the observed signal for free macromolecule in the absence of ligand (F0 , see Section 3.1). Then for the fluorescence quenching experiments have to be Sobs = (F − F0 )/F0 [21]. A horizontal line was drawn intersecting the three titration curves in Fig. 3, defining one constant value of Sobs and three sets of (LTx ;
R6 F = 6 0 F0 R0 + r 6
(9)
where E is the efficiency of energy transfer and R0 is the critical distance when the transfer efficiency is 50%. R06 = 8.79 × 10−25 K 2 n−4 J
(10)
K2
is the orientation space factor, n is the refracted In Eq. (10), index of the medium, is the fluorescence quantum yield of the donor, J is the effect of the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor (see Fig. 4), which may be calculated by the following equation: J=
∞ F()ε()4 d 0 ∞ 0
F()d
(11)
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Fig. 4. Spectral overlaps of the HSA fluorescence (A) with isovitexin absorption (B) ([HSA] = [ISO] = 4.0 M).
where F() is the corrected fluorescence intensity of the donor in the wavelength range from to + , and ε() is the extinction coefficient of the acceptor at . In order to obtain the distance between HSA–Trp214 and ISO, the concentration of both was 4.0 M. In this study, K2 = 2/3, n = 1.36, and = 0.074 [22]. According to Eqs. (9)–(11), it was calculated the values of J = 1.982 × 10−14 cm3 L mol−1 , E = 0.187, R0 = 2.512 nm and r = 3.209 nm. The range distance between donor and acceptor fluorophore is from 2.0 to 8.0 nm [19], indicating that the energy transfer from HSA to ISO likely may occur. 3.6. HSA secondary structural analysis The possible secondary structural changes of HSA induced by binding of ISO were investigated using FT-IR and UV CD spectroscopy analysis. The amide I band is in the region from 1600 to 1700 cm−1 and attributed to the stretching vibration of C O of amide groups in the main chain of the protein. The amide I band of HSA presents centered at
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part in electrostatic interactions (charge neutralization), except the Tyr452 residue which is involved in hydrophobic interactions with the neutral charge distribution at the planar face of ring B of ISO (Fig. S10). Asn295 side chain makes up a hydrogen bond with ISO, ˚ The oxygen atom of exhibiting a formation distance of 2.015 A. Asn295 main chain forms a hydrogen bond with the H6 hydrogen atom of ISO. The polar side chain residues like Lys195, Arg218, Asp451 and Lys436 participate in electrostatic interactions, except the Lys195 residue whose (carbons) nonpolar atoms of the side chain perform hydrophobic interactions with the planar face of the ring B of ISO, as well as Tyr452 residue. Arg218 and Asp451 residues are involved in electrostatic interactions of charge neutralization with the flavonoid glycosylation. A special case occurs with Lys436 residue, it performs electrostatic interactions of the cation– type with the ring B of ISO (see Fig. 6). The cation– interaction is a noncovalent molecular interaction that occurs between a cation (positive anion) and the face of an electron-rich system as the B benzene ring of ISO. Such noncovalent interaction has bonding energies comparable with hydrogen bond energies [26]. 3.9. Accessible superficial area calculation
Fig. 6. (a) Location of isovitexin molecule near the single tryptophan residue (Trp 214) of the HSA in the subdomain IIA (site 1). (b) Structural details of the interaction between HSA and isovitexin obtained by molecular modeling method. Isovitexin molecule is shown as sticks and balls model, the amino acid residues are denoted as sticks model (C, gray; O, red; N, blue; S, yellow; H, white), the –cation interactions are shown as yellow cones, and the hydrogen bonds depicted by green dotted line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.8. Molecular modeling calculations Fig. 6 shows the result of the best binding energy among the molecular modeling simulations. The best binding energy for the HSA–ISO complex is −27.5 kJ mol−1 , with a corresponding theoretical binding constant of 6.7 × 104 M−1 at 298 K, which is in agreement with the values determined by analysis of binding equilibria (Kb , Section 3.2) and binding density function method (Ka , Section 3.4). Fig. 6a shows that ISO is at the entrance of the site 1 of HSA and closes to the Trp214 residue. The entrance of the site 1 of HSA is lesser hydrophobic region than the pocket bottom, as shown at the hydrophobic surface generated for HSA–ISO complex by Chimera 1.8 software [25] (Supplementary material, Fig. S11). Fig. 6b presents in more details the binding site environment. A set of nonpolar side chain residues like Ala191, Pro339, Val343, Pro447, Cys448 and Val455 mostly contributes to the hydrophobic interactions with ISO. However, Ala191 and Pro339 residues form hydrogen bonds with ISO, presenting formation distances ˚ respectively. The oxygen atom of Ala191 of 1.989 and 2.036 A, main chain makes up a hydrogen bond with the H4 hydrogen atom of ISO, this hydrogen atom presents the densest positive charge distribution shown at the MEP of the flavonoid (Fig. S10). The oxygen atom of Pro339 main chain forms a hydrogen bond with the H4 hydrogen atom of ISO. The neutral polar side chain residues like Gln221, Asn295, Tyr341 and Tyr452 are mainly taking
The average area lost by HSA residues in the complexation with ISO was approximately 25 A˚ 2 . Arg218, Lys195 and Tyr452 were the residues with highest loss of ASA in the complex formation (Supplementary material, Table S3). ASA changes for nonpolar and polar atoms of the residues involved in HSA–ISO complexation was of 70% and 30%, respectively. Although, Lys195, Asp451 and Tyr452 are polar residues their largest change in ASA occurred in their nonpolar atoms, demonstrating that these residues participate of hydrophobic interactions in the complexation. ASA results suggest that the hydrophobic interactions are the main interactions involved in the HSA–ISO complex formation even though the electrostatic interaction should not be neglected, which is in accordance with the thermodynamic analysis (Section 3.3). 4. Conclusion The interaction between flavone isovitexin and HSA investigated using a pool of spectroscopic techniques that in combination with ab initio and molecular modeling calculations observed and described in more depth the complex formation. The main physicochemical observable element was the fluorescence signal of the Trp214 residue which was quenched with the addition of ISO aliquots, revealing that ISO molecule is in the vicinity of the Trp214 microenvironment. The validation of the procedure through two different methodologies, Stern–Volmer binding equilibrium and binding density function analysis, both reached similar values of binding constant, number of sites and the energetic of the interaction. These approaches are different with respect to other analytical techniques such as HPLC-UV and HPLC–MS commonly used in pharmacokinetic measurements that rely on some internal standard. The comparison between these two groups of analytical techniques unveils that while HPLC-UV and HPLC–MS can verify the presence of the analyte in the blood plasma. This specific condition could complicate the analysis herewith because is not possible to distinguish the protein in which the observable is (Trp, in our case). Nevertheless, if the protein of interest is purified from the blood plasma and performed analyses with HPLC-UV and HPLC–MS, such techniques could not give details as we demonstrated in the present investigation. Regarding the complex formation the assays with temperature suggested that the quenching mechanism was static, and the same results were also determined carefully by means of UV–vis absorption difference spectra of HSA. The analysis of binding equilibria
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demonstrated that only one ISO molecule binds to HSA, and from the binding constant (Kb ) we calculated the thermodynamic parameters for the formation of the HSA–ISO complex. The negative G values indicated that the binding reaction was spontaneous, and the values of the enthalpy and entropy changes suggested that the hydrophobic interactions played an important role at the formation and stabilization of the HSA–ISO complex. The observation was essentially phenomenological, so that it did not reveal the nature of the hydrophobic forces which results came from the dispersion forces. These forces alone could not explain the complex formation, and this led to the conclusion that the complex assembly was a result of the behavior of the system itself. Those considerations could also be applied to the electrostatic interactions (van der Waals interactions, hydrogen bonds, charge neutralization, cation– interaction). The data obtained from binding density function (BDF) method matched with binding equilibrium analysis; it was an important result since BDF method is independent of any binding model. The distance between donor (HSA–Trp214) and acceptor (ISO) calculated by FRET was 3.209 nm, suggesting that the energy transfer occurred. From FT-IR and CD spectroscopy analysis, no significant change of secondary structure due to the formation of the HSA–ISO complex was observed. The molecular modeling calculations indicated that the ligand as a whole matches its molecular architecture (MEP) with the binding pocket of subdomain IIA of HSA. The results indicated that the interaction of isovitexin with HSA had a contribution balance between electrostatic (hydrogen bonds, charge neutralization and cation– interaction) and hydrophobic interactions, which was also in agreement with the thermodynamic analysis, describing a detailed view of the microenvironment of the binding site. Acknowledgments The author IPC gratefully acknowledges a CAPES scholarship and financial support received from FAPESP. IPC also recognizes GridUNESP by availability of Gaussian 09 quantum chemical program, Prof. Dr. Alexandre Suman de Araújo by feasibility of computational time for AutoDock program in Calix cluster, Prof. Dr. João Ruggiero Neto by availability of spectropolarimeter, and Prof. Dr. Márcio Francisco Colombo by feasibility of UV–vis spectrometer. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.05.015. References [1] E. Grotewold, The Science of Flavonoids, Springer, New York, 2006. [2] S.T. Huang, C.T. Chen, K.T. Chieng, S.H. Huang, B.H. Chiang, L.F. Wang, H.S. Kuo, C.M. Lin, Inhibitory effects of a rice hull constituent on tumor necrosis factor ␣, prostaglandin E2, and cyclooxygenase-2 production in lipopolysaccharideactivated mouse, Ann. N. Y. Acad. Sci. 1042 (2005) 387–395. [3] R.G. Coelho, L.M. Batista, L.C. Santos, A.R.M.S. Brito, W. Vilegas, Phytochemical study and antiulcerogenic activity of Syngonanthus bisulcatus (Eriocaulaceae) Braz, J. Pharm. Sci. 42 (2006) 413–417.
[4] C. Yan, H. Liu, L. Lin, Simultaneous determination of vitexin and isovitexin in rat plasma after oral administration of Santalum album L. leaves extract by liquid chromatography tandem mass spectrometry, Biomed. Chromatogr. 27 (2013) 228–232. [5] T. Peters, All About Albumin. Biochemistry, Genetics and Medical Application, Academic Press, San Diego, CA, 1996. [6] I.P. Caruso, W. Vilegas, M.A. Fossey, M.L. Cornélio, Exploring the binding mechanism of Guaijaverin to human serum albumin: fluorescence spectroscopy and computational approach, Spectrochim. Acta A 97 (2012) 449–455. [7] L. Pohjala, P. Tammela, Aggregating behavior of phenolic compounds – a source of false bioassay results? Molecules 17 (2012) 10774–10790. [8] I.E. Borissevitch, More about the inner filter effect: corrections of Stern–Volmer fluorescence quenching constants are necessary at very low optical absorption of the quencher, J. Lumin. 81 (1999) 219–224. [9] Y.N. Chirgadze, O.V. Fedorov, N.P. Trushina, Estimation of amino acid residue side-chain absorption in the infrared spectra of protein solutions in heavy water, Biopolymers 14 (1975) 679–694. [10] D.M. Byler, H. Susi, Examination of the secondary structure of protein by deconvolved FTIR spectra, Biopolymers 25 (1986) 469–487. [11] N. Sreerama, R.W. Woody, A self-consistent method for the analysis of protein secondary structure from circular dichroism, Anal. Biochem. 282 (2000) 252–260. [12] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision A.02, Gaussian, Inc., Wallingford, CT, 2009. [13] E. Cances, B. Mennucci, J. Tomasi, Evaluation of solvent effects in isotropic and anisotropic dielectrics, and in ionic solutions with a unified integral equation method: theoretical bases, computational implementation and numerical applications, J. Chem. Phys. 107 (1997) 3032–3041. [14] S. Sugio, A. Kashima, S. Mochikuzi, M. Noda, K. Kobayashi, Crystal structure of human serum albumin at 2.5 A˚ resolution, Protein Eng. 12 (1999) 439–446. [15] M.F. Sanner, Python: a programming language for software integration and development, J. Mol. Graphics Modell. 17 (1999) 57–61. [16] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, Automated docking using a Larmarckian genetic algorithm and an empirical binding free energy function, J. Comput. Chem. 19 (1998) 1639–1662. [17] S.J. Hubbard, J.M. Thornton, NACCESS; Computer Program, Department of Biochemistry and Molecular Biology, University College, London, 1993. [18] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer Academic Publishers/Plenum Press, New York, 1999. [19] S. Bi, L. Ding, Y. Tian, D. Song, X. Zhou, X. Liu, H. Zhang, Investigation of the interaction between flavonoids and human serum albumin, J. Mol. Struct. 703 (2004) 37–45. [20] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20 (1981) 3096–3102. [21] T.M. Lohman, W. Bujalowski, Thermodynamics methods for modelindependent determination of equilibrium binding isotherms for protein–DNA interactions: spectroscopic approaches to monitor binding, Methods Enzymol. 208 (1991) 158–290. [22] Y.J. Hu, Y. Liu, J.B. Wang, X.H. Xiao, S.S. Qu, Study of the interaction between monoammonium glycyrrhizinate and bovine serum albumin, J. Pharm. Biomed. Anal. 36 (2004) 915–919. [23] A. Barth, Infrared spectroscopy of proteins, Biochim. Biophys. Acta 1767 (2007) 1073–1101. [24] M.G. Gore, Spectrophotometry and Spectrofluorimetry: A Practical Approach, Oxford University Press, New York, 2000, pp. 123–125. [25] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E. Ferrin, UCSF Chimera – a visualization system for exploratory research and analysis, J. Comput. Chem. 25 (2004) 1605–1612. [26] J.C. Ma, D.A. Dougherty, The cation– interaction, Chem. Rev. 97 (1997) 1303–1324.
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Binding of antioxidant flavone isovitexin to human serum albumin investigated by experimental and computational assays Ícaro Putinhon Caruso a,b , Wagner Vilegas c , Fátima Perreira de Souza a,b , Marcelo Andrés Fossey a,b , Marinônio Lopes Cornélio a,b,∗ a Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas (IBILCE), UNESP, Rua Cristovão Colombo 2265, CEP 15054-000, São José do Rio Preto, SP, Brazil b Centro Multiusuário de Inovac¸ão Biomolecular (CMIB), Instituto de Biociências, Letras e Ciências Exatas (IBILCE), UNESP, Rua Cristovão Colombo 2265, CEP 15054-000, São José do Rio Preto, SP, Brazil c Instituto de Química (IQ), UNESP, Rua Prof. Francisco Degni 55, CEP 14800-900, Araraquara, SP, Brazil
a r t i c l e
i n f o
Article history: Received 27 January 2014 Received in revised form 30 April 2014 Accepted 13 May 2014 Available online 22 May 2014 Keywords: Isovitexin Human serum albumin Fluorescence spectroscopy Binding density function Molecular modeling
a b s t r a c t The flavonoids are a large class of polyphenolic compounds which occur naturally in plants where they are widely distributed. Isovitexin (ISO) is a glycosylated flavonoid that exhibits a potential antioxidant activity. Some recent studies have shown the pharmacokinetic activity of isovitexin in rat blood plasma, however, without detailing the molecular target that is linked and what physicochemical forces govern the interaction. In mammalians, the most abundant protein in blood plasma is the albumin and is not unlike with human, which human serum albumin (HSA) is the major extracellular protein and functions as a carrier of various drugs. The interaction between HSA and ISO was investigated using fluorescence, UV–vis absorbance, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) together with, computational methods like ab initio and molecular modeling calculation. Fluorescence quenching indicated that ISO location is within the hydrophobic pocket in subdomain IIA (site 1) of HSA, close to the Trp214 residue. The Stern–Volmer quenching constants determined at 288, 298 and 308 K and its dependence on temperature indicated that the quenching mechanism was static. From the analysis of binding equilibrium were determined; the binding site number and binding constants, with the correspondent thermodynamic parameters, H, G and S for HSA–ISO complex. Also, a second binding analysis, binding density function (BDF) method, which is independent of any binding model pre-established obtained similar results. The fluorescence resonance energy transfer estimated the distance between the donor (HSA–Trp214) and acceptor (ISO), while FT-IR and CD spectroscopy measured possible changes of secondary structure at the formation of the HSA–ISO complex. The optimized geometry of isovitexin calculation performed with its ground state by using DFT/B3LYP/6-311+G(d,p) method. The HSA–ISO complex interactions determined by molecular modeling tool corroborated with the thermodynamic analysis from the experimental data. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Flavonoids are a large class of naturally occurring polyphenolic compounds widely distributed in plants. The literature shows that flavonoids can present antioxidant, anticancer, antiviral,
∗ Corresponding author at: Departamento de Física, Instituto de Biociências, Letras e Ciências Exatas (IBILCE), UNESP, Rua Cristovão Colombo 2265, CEP 15054-000, São José do Rio Preto, SP, Brazil. Tel.: +55 17 32212246; fax: +55 17 32212247. E-mail addresses: [email protected] (Í.P. Caruso), [email protected] (W. Vilegas), [email protected] (F.P. de Souza), [email protected] (M.A. Fossey), [email protected], [email protected] (M.L. Cornélio). http://dx.doi.org/10.1016/j.jpba.2014.05.015 0731-7085/© 2014 Elsevier B.V. All rights reserved.
anti-inflammatory and heart disease protective activities [1]. Isovitexin (apigenin-6-C--d-glucopyranoside) is a glycoside flavonoid consisting of the flavone apigenin and the saccharide glucose, as shown in Fig. 1. Like many antioxidants present anti-inflammatory activity, ISO exhibits a potential antioxidant as well; studies have demonstrated that, in inflammatory processes induced by lipopolysaccharide (LPS) in mouse macrophage, this flavone is capable of inhibiting the production and, or release of tumor necrosis factor ␣ (TNF-␣) and prostaglandin E2 (PG2 ) [2]. Studies also have showed that isovitexin presents a possibly antiulcerogenic activity and it could help in peptic ulcer treatment [3]. ISO molecule is found, for example in rice hulls (Oryza sativa L.) [2], “semprevivas chapadeira” (Syngonanthus bisulcatus Rul.) [3] and tanxiang
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HSA and ISO were 4.0 M. The final ethanol concentration in buffer was 0. The negative enthalpic term provided the greatest contribution for G◦ , characterizing an enthalpically driven exothermic reaction. The enthalpy–entropy magnitude balance indicated that the hydrophobic interaction offered the main contribution for the formation and stabilization of the HSA–ISO complex. However, signal values for enthalpy and entropy changes also indicated that the electrostatic interactions could play an important role in complex stabilization by forming interactions as van der Walls, hydrogen bonds and charge neutralization.
Fig. 3. Titrations of isovitexin in HSA solutions, as monitored by fluorescence quenching of the Trp214 residue of HSA for three different protein concentrations at pH 7.0 and 298 K. LT 1 , LT 2 and LT 3 represent the ligand concentration for the constant fractional signal molar change value of three different protein concentration (MT 1 , MT 2 and MT 3 ). The insert corresponds to only three sets of concentration pairs (LT ; i and LF values. MT ) to exemplify the utilization of Eq. (9) obtaining
MTx ) (x = 1, 2 and 3) for which LF and i were constants (see insert in Fig. 3). i values obtained from Eq. (6), it can be build From the LF and the Scatchard plot for the binding between HSA and ISO (Supplementary material, Fig. S5) without using any binding model a priori. Scatchard plot was built using the following equation:
i
LF
= n Ka − Ka
i
(8)
where Ka is the association constant and n is binding site number. The obtained values for Ka and n from Scatchard plot were 2.35 × 105 M−1 and 1.04, respectively, which are in accordance with the results obtained from analysis of binding equilibria (Section 3.2). 3.5. Distance measurement between HSA–Trp214 and isovitexin
3.4. Binding density function method The thermodynamic basis for the binding density function (BDF) of ligands bound per macromolecule method is that the distribution in i different states ( i ) is strictly determined at equilibrium by the free ligand concentration (LF ). Thus, if LF is the same for two (or more) solutions at different total macromolecule concentration i will also be the same in each solution. As a result, (MT ), then constant values of LF and i will exist for a number of different combinations of LT and MT , that satisfy the mass conservation equation [21]:
Fluorescence resonance energy transfer (FRET) is a method that can monitor the proximity and relative angular orientation of fluorophores. According to Förster non-radiative energy transfer theory. The energy transfer occurs under the following conditions. The donor produces fluorescent light, the emission spectrum of the donor and the absorbance spectrum of the acceptor have a partial overlap, and the distance between the donor and acceptor is less than 8.0 nm [18]. According to this theory, the average distance r in the binding between HSA–Trp214 (donor) and ISO (acceptor) can be calculated by the equation:
LT = LF +
E =1−
i MT
(6)
Considering the general relationship between the concentration of each macromolecule species with i ligands bound and the experimentally observed signal (Sobs ) from the macromolecule, the fractional signal molar changes (Sobs ) observed in the presence of LT and MT will be given by: Sobs
S − SF MT = obs SF MT
(7)
where SF MT is the observed signal for free macromolecule in the absence of ligand (F0 , see Section 3.1). Then for the fluorescence quenching experiments have to be Sobs = (F − F0 )/F0 [21]. A horizontal line was drawn intersecting the three titration curves in Fig. 3, defining one constant value of Sobs and three sets of (LTx ;
R6 F = 6 0 F0 R0 + r 6
(9)
where E is the efficiency of energy transfer and R0 is the critical distance when the transfer efficiency is 50%. R06 = 8.79 × 10−25 K 2 n−4 J
(10)
K2
is the orientation space factor, n is the refracted In Eq. (10), index of the medium, is the fluorescence quantum yield of the donor, J is the effect of the spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor (see Fig. 4), which may be calculated by the following equation: J=
∞ F()ε()4 d 0 ∞ 0
F()d
(11)
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Fig. 4. Spectral overlaps of the HSA fluorescence (A) with isovitexin absorption (B) ([HSA] = [ISO] = 4.0 M).
where F() is the corrected fluorescence intensity of the donor in the wavelength range from to + , and ε() is the extinction coefficient of the acceptor at . In order to obtain the distance between HSA–Trp214 and ISO, the concentration of both was 4.0 M. In this study, K2 = 2/3, n = 1.36, and = 0.074 [22]. According to Eqs. (9)–(11), it was calculated the values of J = 1.982 × 10−14 cm3 L mol−1 , E = 0.187, R0 = 2.512 nm and r = 3.209 nm. The range distance between donor and acceptor fluorophore is from 2.0 to 8.0 nm [19], indicating that the energy transfer from HSA to ISO likely may occur. 3.6. HSA secondary structural analysis The possible secondary structural changes of HSA induced by binding of ISO were investigated using FT-IR and UV CD spectroscopy analysis. The amide I band is in the region from 1600 to 1700 cm−1 and attributed to the stretching vibration of C O of amide groups in the main chain of the protein. The amide I band of HSA presents centered at
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part in electrostatic interactions (charge neutralization), except the Tyr452 residue which is involved in hydrophobic interactions with the neutral charge distribution at the planar face of ring B of ISO (Fig. S10). Asn295 side chain makes up a hydrogen bond with ISO, ˚ The oxygen atom of exhibiting a formation distance of 2.015 A. Asn295 main chain forms a hydrogen bond with the H6 hydrogen atom of ISO. The polar side chain residues like Lys195, Arg218, Asp451 and Lys436 participate in electrostatic interactions, except the Lys195 residue whose (carbons) nonpolar atoms of the side chain perform hydrophobic interactions with the planar face of the ring B of ISO, as well as Tyr452 residue. Arg218 and Asp451 residues are involved in electrostatic interactions of charge neutralization with the flavonoid glycosylation. A special case occurs with Lys436 residue, it performs electrostatic interactions of the cation– type with the ring B of ISO (see Fig. 6). The cation– interaction is a noncovalent molecular interaction that occurs between a cation (positive anion) and the face of an electron-rich system as the B benzene ring of ISO. Such noncovalent interaction has bonding energies comparable with hydrogen bond energies [26]. 3.9. Accessible superficial area calculation
Fig. 6. (a) Location of isovitexin molecule near the single tryptophan residue (Trp 214) of the HSA in the subdomain IIA (site 1). (b) Structural details of the interaction between HSA and isovitexin obtained by molecular modeling method. Isovitexin molecule is shown as sticks and balls model, the amino acid residues are denoted as sticks model (C, gray; O, red; N, blue; S, yellow; H, white), the –cation interactions are shown as yellow cones, and the hydrogen bonds depicted by green dotted line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.8. Molecular modeling calculations Fig. 6 shows the result of the best binding energy among the molecular modeling simulations. The best binding energy for the HSA–ISO complex is −27.5 kJ mol−1 , with a corresponding theoretical binding constant of 6.7 × 104 M−1 at 298 K, which is in agreement with the values determined by analysis of binding equilibria (Kb , Section 3.2) and binding density function method (Ka , Section 3.4). Fig. 6a shows that ISO is at the entrance of the site 1 of HSA and closes to the Trp214 residue. The entrance of the site 1 of HSA is lesser hydrophobic region than the pocket bottom, as shown at the hydrophobic surface generated for HSA–ISO complex by Chimera 1.8 software [25] (Supplementary material, Fig. S11). Fig. 6b presents in more details the binding site environment. A set of nonpolar side chain residues like Ala191, Pro339, Val343, Pro447, Cys448 and Val455 mostly contributes to the hydrophobic interactions with ISO. However, Ala191 and Pro339 residues form hydrogen bonds with ISO, presenting formation distances ˚ respectively. The oxygen atom of Ala191 of 1.989 and 2.036 A, main chain makes up a hydrogen bond with the H4 hydrogen atom of ISO, this hydrogen atom presents the densest positive charge distribution shown at the MEP of the flavonoid (Fig. S10). The oxygen atom of Pro339 main chain forms a hydrogen bond with the H4 hydrogen atom of ISO. The neutral polar side chain residues like Gln221, Asn295, Tyr341 and Tyr452 are mainly taking
The average area lost by HSA residues in the complexation with ISO was approximately 25 A˚ 2 . Arg218, Lys195 and Tyr452 were the residues with highest loss of ASA in the complex formation (Supplementary material, Table S3). ASA changes for nonpolar and polar atoms of the residues involved in HSA–ISO complexation was of 70% and 30%, respectively. Although, Lys195, Asp451 and Tyr452 are polar residues their largest change in ASA occurred in their nonpolar atoms, demonstrating that these residues participate of hydrophobic interactions in the complexation. ASA results suggest that the hydrophobic interactions are the main interactions involved in the HSA–ISO complex formation even though the electrostatic interaction should not be neglected, which is in accordance with the thermodynamic analysis (Section 3.3). 4. Conclusion The interaction between flavone isovitexin and HSA investigated using a pool of spectroscopic techniques that in combination with ab initio and molecular modeling calculations observed and described in more depth the complex formation. The main physicochemical observable element was the fluorescence signal of the Trp214 residue which was quenched with the addition of ISO aliquots, revealing that ISO molecule is in the vicinity of the Trp214 microenvironment. The validation of the procedure through two different methodologies, Stern–Volmer binding equilibrium and binding density function analysis, both reached similar values of binding constant, number of sites and the energetic of the interaction. These approaches are different with respect to other analytical techniques such as HPLC-UV and HPLC–MS commonly used in pharmacokinetic measurements that rely on some internal standard. The comparison between these two groups of analytical techniques unveils that while HPLC-UV and HPLC–MS can verify the presence of the analyte in the blood plasma. This specific condition could complicate the analysis herewith because is not possible to distinguish the protein in which the observable is (Trp, in our case). Nevertheless, if the protein of interest is purified from the blood plasma and performed analyses with HPLC-UV and HPLC–MS, such techniques could not give details as we demonstrated in the present investigation. Regarding the complex formation the assays with temperature suggested that the quenching mechanism was static, and the same results were also determined carefully by means of UV–vis absorption difference spectra of HSA. The analysis of binding equilibria
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demonstrated that only one ISO molecule binds to HSA, and from the binding constant (Kb ) we calculated the thermodynamic parameters for the formation of the HSA–ISO complex. The negative G values indicated that the binding reaction was spontaneous, and the values of the enthalpy and entropy changes suggested that the hydrophobic interactions played an important role at the formation and stabilization of the HSA–ISO complex. The observation was essentially phenomenological, so that it did not reveal the nature of the hydrophobic forces which results came from the dispersion forces. These forces alone could not explain the complex formation, and this led to the conclusion that the complex assembly was a result of the behavior of the system itself. Those considerations could also be applied to the electrostatic interactions (van der Waals interactions, hydrogen bonds, charge neutralization, cation– interaction). The data obtained from binding density function (BDF) method matched with binding equilibrium analysis; it was an important result since BDF method is independent of any binding model. The distance between donor (HSA–Trp214) and acceptor (ISO) calculated by FRET was 3.209 nm, suggesting that the energy transfer occurred. From FT-IR and CD spectroscopy analysis, no significant change of secondary structure due to the formation of the HSA–ISO complex was observed. The molecular modeling calculations indicated that the ligand as a whole matches its molecular architecture (MEP) with the binding pocket of subdomain IIA of HSA. The results indicated that the interaction of isovitexin with HSA had a contribution balance between electrostatic (hydrogen bonds, charge neutralization and cation– interaction) and hydrophobic interactions, which was also in agreement with the thermodynamic analysis, describing a detailed view of the microenvironment of the binding site. Acknowledgments The author IPC gratefully acknowledges a CAPES scholarship and financial support received from FAPESP. IPC also recognizes GridUNESP by availability of Gaussian 09 quantum chemical program, Prof. Dr. Alexandre Suman de Araújo by feasibility of computational time for AutoDock program in Calix cluster, Prof. Dr. João Ruggiero Neto by availability of spectropolarimeter, and Prof. Dr. Márcio Francisco Colombo by feasibility of UV–vis spectrometer. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.05.015. References [1] E. Grotewold, The Science of Flavonoids, Springer, New York, 2006. [2] S.T. Huang, C.T. Chen, K.T. Chieng, S.H. Huang, B.H. Chiang, L.F. Wang, H.S. Kuo, C.M. Lin, Inhibitory effects of a rice hull constituent on tumor necrosis factor ␣, prostaglandin E2, and cyclooxygenase-2 production in lipopolysaccharideactivated mouse, Ann. N. Y. Acad. Sci. 1042 (2005) 387–395. [3] R.G. Coelho, L.M. Batista, L.C. Santos, A.R.M.S. Brito, W. Vilegas, Phytochemical study and antiulcerogenic activity of Syngonanthus bisulcatus (Eriocaulaceae) Braz, J. Pharm. Sci. 42 (2006) 413–417.
[4] C. Yan, H. Liu, L. Lin, Simultaneous determination of vitexin and isovitexin in rat plasma after oral administration of Santalum album L. leaves extract by liquid chromatography tandem mass spectrometry, Biomed. Chromatogr. 27 (2013) 228–232. [5] T. Peters, All About Albumin. Biochemistry, Genetics and Medical Application, Academic Press, San Diego, CA, 1996. [6] I.P. Caruso, W. Vilegas, M.A. Fossey, M.L. Cornélio, Exploring the binding mechanism of Guaijaverin to human serum albumin: fluorescence spectroscopy and computational approach, Spectrochim. Acta A 97 (2012) 449–455. [7] L. Pohjala, P. Tammela, Aggregating behavior of phenolic compounds – a source of false bioassay results? Molecules 17 (2012) 10774–10790. [8] I.E. Borissevitch, More about the inner filter effect: corrections of Stern–Volmer fluorescence quenching constants are necessary at very low optical absorption of the quencher, J. Lumin. 81 (1999) 219–224. [9] Y.N. Chirgadze, O.V. Fedorov, N.P. Trushina, Estimation of amino acid residue side-chain absorption in the infrared spectra of protein solutions in heavy water, Biopolymers 14 (1975) 679–694. [10] D.M. Byler, H. Susi, Examination of the secondary structure of protein by deconvolved FTIR spectra, Biopolymers 25 (1986) 469–487. [11] N. Sreerama, R.W. Woody, A self-consistent method for the analysis of protein secondary structure from circular dichroism, Anal. Biochem. 282 (2000) 252–260. [12] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery Jr., J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, O. Farkas, J.B. Foresman, J.V. Ortiz, J. Cioslowski, D.J. Fox, Gaussian 09, Revision A.02, Gaussian, Inc., Wallingford, CT, 2009. [13] E. Cances, B. Mennucci, J. Tomasi, Evaluation of solvent effects in isotropic and anisotropic dielectrics, and in ionic solutions with a unified integral equation method: theoretical bases, computational implementation and numerical applications, J. Chem. Phys. 107 (1997) 3032–3041. [14] S. Sugio, A. Kashima, S. Mochikuzi, M. Noda, K. Kobayashi, Crystal structure of human serum albumin at 2.5 A˚ resolution, Protein Eng. 12 (1999) 439–446. [15] M.F. Sanner, Python: a programming language for software integration and development, J. Mol. Graphics Modell. 17 (1999) 57–61. [16] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson, Automated docking using a Larmarckian genetic algorithm and an empirical binding free energy function, J. Comput. Chem. 19 (1998) 1639–1662. [17] S.J. Hubbard, J.M. Thornton, NACCESS; Computer Program, Department of Biochemistry and Molecular Biology, University College, London, 1993. [18] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer Academic Publishers/Plenum Press, New York, 1999. [19] S. Bi, L. Ding, Y. Tian, D. Song, X. Zhou, X. Liu, H. Zhang, Investigation of the interaction between flavonoids and human serum albumin, J. Mol. Struct. 703 (2004) 37–45. [20] P.D. Ross, S. Subramanian, Thermodynamics of protein association reactions: forces contributing to stability, Biochemistry 20 (1981) 3096–3102. [21] T.M. Lohman, W. Bujalowski, Thermodynamics methods for modelindependent determination of equilibrium binding isotherms for protein–DNA interactions: spectroscopic approaches to monitor binding, Methods Enzymol. 208 (1991) 158–290. [22] Y.J. Hu, Y. Liu, J.B. Wang, X.H. Xiao, S.S. Qu, Study of the interaction between monoammonium glycyrrhizinate and bovine serum albumin, J. Pharm. Biomed. Anal. 36 (2004) 915–919. [23] A. Barth, Infrared spectroscopy of proteins, Biochim. Biophys. Acta 1767 (2007) 1073–1101. [24] M.G. Gore, Spectrophotometry and Spectrofluorimetry: A Practical Approach, Oxford University Press, New York, 2000, pp. 123–125. [25] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E. Ferrin, UCSF Chimera – a visualization system for exploratory research and analysis, J. Comput. Chem. 25 (2004) 1605–1612. [26] J.C. Ma, D.A. Dougherty, The cation– interaction, Chem. Rev. 97 (1997) 1303–1324.
